K-ASSAY 
KAMIYA BIOMEDICAL COMPANY
KAMIYA BIOMEDICAL COMPANY
Hemoglobin Colorimetric
Detection kit
For the quantitative determination of hemoglobin in
whole blood, RBCs, and hemolyzed serum and plasma
Cat. No. KT-731
For Research Use Only.
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KAMIYA BIOMEDICAL COMPANY
PRODUCT INFORMATION
Hemoglobin Colorimetric Detection kit
Cat. No. KT-731
BACKGROUND
Hemoglobin (Hgb) is an erythrocyte protein complex comprised of two sets of identical pairs of subunits, each of which bind
an iron-prophyrin group commonly called heme. Generally containing two alpha or alpha-like globulin chains, the remaining
subunits may be beta, gamma, delta or epsilon, or in the case of infants, fetal hemoglobin that is replaced during the first
year of life. Heme binds and releases oxygen or carbon dioxide in response to slight changes in local gas tension. Free
oxygen or carbon dioxide bound by one heme group facilitates subsequent binding by the other heme groups in a given
hemoglobin molecule. Subtle changes in pH also regulate hemoglobin affinity for free gases, resulting in a high level of
hemostatic control. Hemoglobin values are associated with a variety of conditions ranging from anemias (low Hgb),
erythrocytosis (high Hgb), thalassemias (aberrant chain synthesis), and sickling disorders (abnormal complex shape).
The universal reference procedure for hemoglobin determination in blood has been the cyanmethemoblobin method as
determined by the Clinical and Laboratory Standards Institute and the International Council for Standardization in
Haematology. In this method, ferricyanide and potassium cyanide convert hemoglobin to a more stable cyanmethemoglobin
form that is measured photometrically. While this method is straightforward and uses a single reaction solution, not all forms
of hemoglobin are converted to cyanmethemoglobin at the same rate or even to completion. In addition to the safety issues
surrounding cyanide, the reagent itself is not stable, so extra care needs to be taken to ensure the quality of any
measurement.
PRINCIPLE
The Hemoglobin detection kit is designed to quantitatively measure all forms of hemoglobin present in blood and RBCs, or
plasma and serum. Please read the complete kit insert before performing this assay. A human hemoglobin calibrator is
provided to generate a calibration curve for the assay and all samples should be read off the calibration curve. Calibrators or
diluted samples are pipetted into a clear microtiter plate and the ready-to-use Hemoglobin Detection Reagent is added to
each well. For whole blood or RBC samples 10 µL of samples and calibrators are used in the Regular format, for serum and
plasma samples 100 µL are used in the High Sensitivity format. Results are calculated as g/dL for whole blood and RBCs,
and mg/mL for serum and plasma. The plate is incubated for 30 minutes at room temperature. The plate is read at 560-580
nm to detect the intensity of the color generated. The concentration of the hemoglobin in the sample is calculated, after
making suitable correction for dilution, using software available with most plate readers.
The Hemoglobin Detection kit uses a single reaction solution that is light stable at 4°C and does not contain dangerous
chemicals. All forms of hemoglobin are rapidly converted to a single stable form that is measured photometrically. Many
samples can be measured without dilution in this safe, simple assay.
COMPONENTS
Clear 96 Well Plates Two plates
Hemoglobin Calibrator 300 µL
A stock solution of human hemoglobin at 16 g/dL.
Hemoglobin Sample Diluent 50 mL
Sample diluent containing detergent and ≤ 0.09% sodium azide.
Hemoglobin Detection Reagent 20 mL
A solution containing chemicals that react with hemoglobin.
Caution: Caustic.
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STORAGE
All components of this kit should be stored at 4°C until the expiration date of the kit.
OTHER MATERIALS REQUIRED
Repeater pipet with disposable tips capable of dispensing 100 µL.
Colorimetric 96 well microplate reader capable of reading optical density at between 560 and 580 nm. Please see spectra of
reaction below:
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic
curve (4PLC) fitting. Contact your plate reader manufacturer for details.
PRECAUTIONS
As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction. The
complete insert should be read and understood before attempting to use the product.
The Hemoglobin Calibrator is derived from human blood. It has been extensively tested for viral contamination, but all
human blood products should be treated as potentially infectious and adequate precautions taken.
The Hemoglobin Detection Reagent is basic. The solution should not come in contact with skin or eyes. Take appropriate
safety precautions when handling this reagent.
Some components of the kit contain sodium azide, which may react with lead or copper plumbing to form potentially
explosive complexes. When disposing of reagents always flush with large volumes of water to prevent azide build-up.
SAMPLE TYPES
This assay has been validated for whole blood, and hemolyzed serum, EDTA and heparin plasma samples from multiple
species, including whole blood and RBCs from human, chicken and dogfish. Serum and plasma samples from human,
mouse, rabbit and sheep samples were also tested. Samples containing visible particulate should be centrifuged prior to
using.
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Bright yellow colored samples may interfere with the High Sensitivity format and may require blanking prior to addition of the
Detection Reagent. Blanking of brightly colored samples is carried out by adding the sample or diluted sample to the plate
and reading the optical density at 560-580 nm before the addition of the detection reagent. The optical density from this
blanking step should be subtracted from the optical density for the samples measured.
SAMPLE PREPARATION
For Regular Format
Whole Blood
Whole blood must be diluted ≥ 1:2 with Hemoglobin Sample Diluent prior to running in the kit.
Red Blood Cell/Erythrocytes
RBC samples should be lysed with Hemoglobin Sample Diluent prior to running in the kit.
For High Sensitivity Format
Serum and Plasma
Serum and plasma samples should be run in the High Sensitivity format without any dilution. Hemolyzed samples can be
read in the Regular format.
Any samples with hemoglobin concentrations above the calibration curve range should be diluted further with Hemoglobin
Sample Diluent to obtain readings within the calibration curve.
Use all samples within 2 hours of dilution.
REAGENT PREPARATION
Calibrator Preparation - Regular Format
Label six glass test tubes as #2 through #7. Briefly vortex and spin the vial of calibrator in a microcentrifuge to ensure
contents are at bottom of vial. The Hemoglobin Calibrator supplied in the kit is calibrator 1. Pipet 50 µL of Hemoglobin
Sample Diluent into tubes #2 to #7. Carefully add 50 µL of the Hemoglobin Calibrator in the Calibrator vial provided to tube
#2 and vortex completely. Take 50 µL of the Hemoglobin solution in tube #2 and add it to tube #3 and vortex completely.
Repeat the serial dilutions for tubes #4 through #7. The concentration of Hemoglobin in the Hemoglobin Calibrator vial and
tubes #2 through #7 will be 16, 8, 4, 2, 1, 0.5 and 0.25 g/dL.
Calibrator Preparation - High Sensitivity Format
Label seven glass test tubes as #1 through #7. Briefly vortex and spin the vial of calibrator in a microcentrifuge to ensure
contents are at bottom of vial. Pipet 525 µL of Hemoglobin Sample Diluent into tube #1, and 250 µL into tubes #2 to #7.
Carefully add 75 µL of the Hemoglobin Calibrator provided to tube #1 and vortex completely. Take 250 µL of the Hemoglobin
solution in tube #1 and add it to tube #2 and vortex completely. Repeat the serial dilutions for tubes #3 through #7. The concentration of Hemoglobin in tubes #1 through #7 will be 20, 10, 5, 2.5, 1.25, 0.625 and 0.313 mg/mL.
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Use all Calibrators within 2 hours of preparation.
ASSAY PROTOCOL
Allow the kit reagents to come to room temperature for 30 minutes. We recommend that all calibrators and samples be run
in duplicate to allow the end user to accurately determine hemoglobin concentration. Ensure that all samples have reached
room temperature and have been diluted as appropriate prior to running them in the kit.
Regular Format
1. Use the plate layout sheet on the back page of the insert to aid in proper sample and calibrator identification.
2. Pipet 10 µL of samples or calibrators into wells in the plate. Pipet 10 µL of Hemoglobin Sample Diluent into the zero
calibrator wells.
3. Add 100 µL of the Hemoglobin Detection Reagent to each well, using a repeater pipet. Tap the plate to mix.
4. Incubate at room temperature for 30 minutes.
5. Read the optical density generated from each well in a plate reader capable of reading at 560-580 nm. See spectra for
details.
6. Use the plate reader’s built-in 4PLC software capabilities to calculate Hemoglobin concentration for each sample.
High Sensitivity Format
1. Use the plate layout sheet on the back page of the insert to aid in proper sample and calibrator identification.
2. Pipet 100 µL of samples or calibrators into wells in the plate. Pipet 100 µL of Hemoglobin Sample Diluent into the zero
calibrator wells.
3. Add 100 µL of the Hemoglobin Detection Reagent to each well, using a repeater pipet. Tap the plate to mix.
4. Incubate at room temperature for 30 minutes.
5. Read the optical density generated from each well in a plate reader capable of reading at 560-580 nm. See spectra for
details.
6. Use the plate reader’s built-in 4PLC software capabilities to calculate Hemoglobin concentration for each sample.
CALCULATION OF RESULTS
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Average the duplicate OD readings for each calibrator and sample. Create a calibration curve by reducing the data using the
4PLC fitting routine on the plate reader, after subtracting the mean OD’s for the Zero calibrator. The sample concentrations
obtained should be multiplied by the dilution factor to obtain neat values.
TYPICAL DATA - REGULAR FORMAT
TYPICAL DATA - HIGH SENSITIVTY FORMAT
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Always run your own calibration curve for calculation of results. Do not use this data.
Typical Calibration Curves
Always run your own calibration curves for calculation of results. Do not use this data.
VALIDATION DATA
Sensitivity and Limit of Detection
Sensitivity was calculated by comparing the OD’s for twenty wells run for each of the zero and calibrator #7. The detection
limit was determined at two (2) standard deviations from the zero along the calibration curve.
Sensitivity was determined as 0.021 g/dL for the Regular format and 0.020 mg/mL (0.0020 g/dL) for the High
Sensitivity format.
The Limit of Detection for the assay was determined in a similar manner by comparing the OD’s for twenty replicates for
each of the zero calibrator and a low concentration diluted human sample.
Limit of Detection was determined as 0.021 g/dL for the Regular format and 0.033 mg/mL (0.0033 g/dL) for the High
Sensitivity format.
Linearity
Linearity was determined by taking a human EDTA plasma sample with a low Hgb level of 0.46 g/dL diluted 1:2 with water
and a RBC spiked sheep serum with a high level of 9.45 g/dL diluted 1:2 with water and mixing them in the ratios given
below. The measured concentrations were compared to the expected values based on the ratios used.
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Intra Assay Precision
Three mammalian samples were diluted with Hemoglobin Sample Diluent and run in replicates of 20 in an assay. The mean
and precision of the calculated hemoglobin concentrations were:
Inter Assay Precision
Three mammalian samples were diluted with Hemoglobin Sample Diluent and run in duplicates in ten assays run over
multiple days by three operators. The mean and precision of the calculated hemoglobin concentrations were:
SAMPLE VALUES
This assay has been tested with whole blood, hemolyzed serum, EDTA and heparin plasma samples from multiple species,
including whole blood and RBCs from human, chicken and dogfish. Serum and plasma samples from human, mouse, rabbit
and sheep samples were also tested. Five human whole blood and four human erythrocyte lysates were tested in the assay.
Whole blood values ranged from 13.95 to 21.36 g/dL with an average of 15.77 g/dL and erythrocyte lysates ranged from
21.58 to 40.21 g/dL with and average of 32.23 g/dL, not corrected for hemocrits. Normal reference range for human whole
blood is 12.0 – 17.0 g/dL.
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INTERFERENTS
A whole blood sample was serially diluted with 40 g/dL BSA to test for protein interference and tested in the assay. No
significant change in the measured hemoglobin level was observed over five two-fold dilutions.
To test for glucose interference a whole blood sample was serially diluted with 2 g/dL glucose and tested in the assay. No
significant change in the measured hemoglobin level was observed over five two-fold dilutions.
For lipid interference a whole blood sample was serially diluted with a mixture containing 0.8 g/dL cholesterol and 11.2 g/dL
triglycerides and tested in the assay. No significant change in the measured hemoglobin level was observed over five
two-fold dilutions.
A whole blood sample was serially diluted with 2 mg/dL of bilirubin and tested in the assay. A 0.7% change in the measured
hemoglobin level was observed. Bilirubin at 2 mg/dL in normal adults would be considered jaundiced. Newborns can have
bilirubin levels above 5 mg/dL.
FOR RESEARCH USE ONLY
KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068
Fax: (206) 575-8094
Email: [email protected]
www.k-assay.com
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